Production of cheese and cultured dairy products has long relied on the fermentation of milk by group N streptococci. Members of this group, composed of Streptococcus lactis, S. cremoris, and S. lactis subsp. diacetylactis, are directly responsible for the acid development, flavor production, and often coagulum characteristics in mesophilic dairy fermentations. Because efficient milk fermentations are dependent on the growth and activity of the lactic streptococci, great care is exercised to prepare starter cultures that are highly active and uncontaminated with undesirable microorganisms or bacteriophages. However, the fermentation process itself is nonaseptic, occurring in open vats with a nonsterile medium, pasteurized milk. It is therefore highly susceptible to contamination with bacteriophages. For the majority of strains of lactic streptococci employed in commercial dairy fermentations, lytic bacteriophages capable of halting growth and acid production can appear within one to two days after introducing the culture into the cheese plant. Although bacteriophage contamination of numerous industrial fermentations has been observed, the destructive role of bacteriophages in milk fermentations is without parallel in other fermentation processes.
Historically, milk fermentations relied on starter cultures composed of undefined mixtures of lactic streptococci propagated without knowledge of, or protection from, bacteriophages. Natural phage contamination in these cultures established an equilibrium of evolving bacteriophages and phage-resistant variants. These cultures were highly variable in day-to-day levels of acid production, but remained moderately active and could be used continuously in small fermentation factories. Over the past 20 years, starter culture failures due to bacteriophage infection have become prevalent throughout the dairy industry. Increasing demand for cultured milk products in recent years has necessitated increases in both production capacity and process efficiency such that larger volumes of milk are processed, cheese vats are filled repeatedly within a single day, and total processing time is shortened. This modernization of the industry concurrently increased the probability of phage contamination and further dictated the use of defined mixtures of lactic streptococci capable of uniform and rapid rates of acid production. With the selection of highly fermentative lactic streptococci and their propagation under aseptic conditions (in the absence of bacteriophages), the majority of cultures now used by the industry have become highly susceptible to bacteriophage attack upon introduction into the cheese factory.
To cope with bacteriophage problems a number of successful methods have been developed to minimize phage action during commercial milk fermentations. Through the use of concentrated cultures, aseptic bulk starter vessels and phage-inhibitory media (see, for example, U.S. Pat. No. 4,282,255), the starter culture can be protected from bacteriophage infection prior to vat inoculation. However, phage contamination cannot be prevented following entrance into the fermentation vat. Therefore, emphasis for protection of the culture shifts to minimizing prolific phage-host interactions through rotation of phage-unrelated strains or use of phage-resistant mutants in multiple-strain starters. Although, in theory, strain rotation should minimize developing phage populations within the plant, in practice it has proved difficult to identify strains that demonstrate completely different patterns of phage sensitivity. Estimates of the total number of different, phage-unrelated lactic streptococci approximate 25 strains worldwide. Considering the small number of phage-unrelated strains available, the choice of strains for incorporation into rotation programs is severely limited. Similarly, few phage-unrelated strains are available for construction of multiple-strain starters containing composites of four to six strains.
A decade ago, Sandine, W. E., et al., J. Milk Food Technol. 35, 176 (1972) emphasized the need to isolate new strains of lactic streptococci for use in the dairy industry. Foremost among the criteria for selection of these strains was resistance to existing bacteriophages. It is now recognized that some strains of lactic streptococci are not attacked by any phage when challenged with large collections of laboratory phage banks, or when used on a continuous, long-term basis in commercial fermentations. These reports demonstrate the existence of lactic streptococci that are not sensitive to bacteriophage attack, in spite of devastating phage pressure such as that which routinely occurs within the factory environment. However, to date, only a limited number of phage-insensitive strains have been identified and studied for mechanism of phage resistance.
Streptococcus lactis ME2 has been shown to exhibit at least three independent phage defense mechanisms that functioned cooperatively to confer an apparent phage-insensitive state. Sanders, M. E. and T. R. Klaenhammer, Appl Environ. Microbiol. 46, 1125 (1983); Sanders, M. E. and T. R. Klaenhammer, Appl. Environ. Microbiol., 47, 979 (1984). Inhibition of bacteriophage by ME2 was initially characterized to include the following reactions: (i) phage adsorption was retarded in the presence of a 30 Md plasmid, pME0030; (ii) restriction and modification activities were exhibited; and (iii) a heat-sensitive inhibition of phage burst size occurred for modified phage propagated on the adsorption variant, S. lactis N1. Subsequent characterization of S. lactis ME2 exconjugants identified a 30 Md conjugative plasmid (pTR2030) that imposed either a heat-sensitive reduction in burst size of prolate phages without altering the plaquing efficiency, Klaenhammer, T. R. and R. B. Sanozky, J. Gen. Microbiol. 131, 1531 (1985), or a complete elimination of the plaquing ability of small isometric phages without affecting the level of phage adsorption, Jarvis, A. W. and T. R. Klaenhammer, Appl. Environ. Microbiol. 51, 1272 (1986); Steenson, L. R. and T. R. Klaenhammer, Appl. Environ. Microbiol. 50, 851 (1985). Introduction of pTR2030 to phage-sensitive lactic streptococci provided effective protection against small isometric phages in general, without altering the fermentative ability of the strains constructed, Sing, W. D. and T. R. Klaenhammer, Appl. Environ. Microbiol. 51, 1264 (1986). Host dependent phage replication could not be demonstrated on pTR2030 transconjugants, providing evidence that the mechanism of pTR2030-induced resistance did not involve phage restriction and modification activities. Klaenhammer and Sanozky, supra.
Plasmids encoding mechanisms for phage restriction and modification activity appear widely distributed throughout group N streptococci and can be effective in inhibiting heterologous phage attack, depending on the level of phage restriction imposed. Boussemaer, J. P. et al., J. Dairy Res. 47, 401 (1980); Klaenhammer, T. R., Adv. Appl. Microbiol. 30, 1 (1984); Pearce, L. E., N.Z.J. Dairy Sci. Technol. 13, 166 (1978). Host-dependent phage replication has been correlated to the presence of a 10 Md plasmid in S. cremoris KH. Sanders, M. E. and T. R. Klaenhammer, Appl. Environ. Microbiol. 42, 944 (1981).
Chopin, A. et al., Plasmid 11, 260 (1984) report the conjugal transfer of 28 and 31 kilobase plasmids responsible for restriction and modification of phage in lactic streptococci, and suggested that interactions between these and other plasmids affected the level of phage restriction. The plasmids were found in Streptococcus lactis strain IL 594. It was not indicated whether or not the plasmids carried their own transfer determinants.
Hershberger, C. L., U.S. Pat. No. 4,530,904, discloses a method for protecting bacteria in general from different types of bacteriophage. The method involves transforming a bacterium with a recombinant DNA cloning vector. The recombinant vector comprises a replicon that is functional in the bacterium, a gene that expresses a functional polypeptide (i.e., human growth hormone) in the bacterium, and a DNA segment which confers restriction and modification activity to the bacterium. The transformed bacterium is then cultured under large-scale fermentation conditions. This method is particularly adapted to fermentation procedures for the production of polypeptide products like growth hormone.
The identification or creation of plasmids encoding for restriction and modification activity in group N streptococci is necessary in order to genetically engineer strains that meet industrial criteria for fermentative capabilities and long-term phage resistance. The present invention provides for a plasmid which confers phage restriction and modification activity to group N streptococci. Group N streptococci containing the plasmid or a derivative thereof are useful for formulating starter cultures which can be used for the production of cheese and cultured dairy products.